Standards in Genomic Sciences (2014) 9:893-901
DOI:10.4056/sigs.5229330
Complete genome sequence of Thalassolituus oleivorans R6-15, an obligate hydrocarbonoclastic marine bacterium from the Arctic Ocean Chunming Dong1, 2, 3†, Xin Chen1, 2, 3, 4†, Yanrong Xie1, 2, 3, 4, Qiliang Lai1, 2, 3, Zongze Shao1,2, 3* Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, China; 2 State Key Laboratory Breeding Base of Marine Genetic Resources, Xiamen, China; 3 Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen, China; . 4 Life Science College, Xiamen University, Xiamen 361005, Fujian, China. 1
Correspondence: Zongze Shao ([email protected])
*
†
Authors contributed equally to this work.
Keywords: Thalassolituus, genome, alkane-degrading, surface seawater, Arctic Ocean Strain R6-15 belongs to the genus Thalassolituus, in the family Oceanospirillaceae of Gammaproteobacteria. Representatives of this genus are known to be the obligate hydrocarbonoclastic marine bacteria. Thalassolituus oleivorans R6-15 is of special interest due to its dominance in the crude oildegrading consortia enriched from the surface seawater of the Arctic Ocean. Here we describe the complete genome sequence and annotation of this strain, together with its phenotypic characteristics. The genome with size of 3,764,053 bp comprises one chromosome without any plasmids, and contains 3,372 protein-coding and 61 RNA genes, including 12 rRNA genes.
Introduction
Thalassolituus spp. belong to the Oceanospirillaceae of Gammaproteobacteria. The genus was first described by Yakimov et.al. (2004), and is currently composed of two type species, T. oleivorans and T. marinus [1,2]. Bacteria of this genus are known as obligate hydrocarbonoclastic marine bacteria [3]. Previous reports showed that Thalassolituusrelated species were among the most dominant members of the petroleum hydrocarbon-enriched consortia at low temperature [4-7]. In addition to consortia enriched with oil, Thalassolituus spp. can be detected in variety of cold environments as well [8-10]. Strain R6-15 was isolated from the surface seawater of the Arctic Ocean after enriched with crude oil during the fourth Chinese National Arctic Research Expedition of the “Xulong” icebreaker in the summer of 2010. The 16S rRNA gene sequence shared 99.86% and 96.39% similarities with T. oleivorans MIL-1T and T. marinus IMCC1826T, respectively. Pyrosequencing results (16S rRNA gene V3 region) of fifteen oil-degrading consortia across the Arctic Ocean showed that the dominant
member in most of the consortia shared identical sequence of this strain, comprising 8.4-99.6% of the total reads (not published). Here, we described the complete genome sequence and annotation of strain T. oleivorans R615, and its phenotypic characteristics. Moreover, a brief comparison was made between strain R6-15 and the two type strains of the validly named species of this genus, in both phenotypic and genomic aspects.
Classification and features
T. oleivorans R6-15 is closely related with T. oleivorans MIL-1T (Figure 1, Table 1). The strain is aerobic, Gram-negative and motile by a single polar flagellum, exhibiting a characteristic morphology of a curved rod-shape cell (Figure 2). Strain R6-15 is able to utilize a restricted spectrum of carbon substrates for growth, including sodium acetate, Tween-40, Tween-80 and C12-C36 aliphatic hydrocarbons. Its growth temperature ranges from 4 to 32°C with optimum of 25°C.
The Genomic Standards Consortium
Thalassolituus oleivorans 10 100
Thalassolituus oleivorans MIL-1T (AJ431699) Thalassolituus oleivorans R6-15 (KF836058)
89
Oceanobacter kriegii IFO 15467T (AB006767) Bermanella marisrubri RED65T (AY136131)
95
Marinomonas blandensis MED 121 (DQ403809) Marinomonas mediterranea MMB-1T (AF063027)
100
Marinomonas sp. MWYL1 (NR_074778)
85 6 100
Marinomonas posidonica IVIA-Po-181T (EU188445) Oceanospirillum beijerinckii IFO 15445T (AB006760) Oceanospirillum maris IFO 15468T (AB006763)
Neptunomonas japonica JAMM 0745T (AB288092) Amphritea japonica JAMM 1866T (AB330881)
63 7
Neptuniibacter caesariensis MED92T (AY136116) Marinospirillum minutulum ATCC 19193T 0.0
Figure 1. Phylogenetic tree highlighting the position of T. oleivorans strain R6-15 relative to other type and nontype strains with finished or non-contiguous finished genome sequences within the family Oceanospirillaceae. Accession numbers of 16S rRNA gene sequences are indicated in brackets. Sequences were aligned using DNAMAN version 6.0, and a neighbor-joining tree obtained using the maximum-likelihood method within the MEGA version 5.0 [11]. Numbers adjacent to the branches represent percentage bootstrap values based on 1,000 replicates.
Figure 2. Transmission electron micrograph of T. oleivorans R6-15, using a JEM-1230 (JEOL) at an operating voltage of 120 kV. The scale bar represents 0.5 µm. 894
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Dong et al. Table 1. Classification and general features of T. oleivorans R6-15 according to the MIGS recommendations [12]. MIGS ID Property Term Evidence codea Domain Bacteria TAS [13] Phylum Proteobacteria TAS [14] Class Gammaproteobacteria TAS [15-17] Order Oceanospirillales TAS [16,18] Current classification Family Oceanospirillaceae TAS [16,19] Genus Thalassolituus TAS [1] Species Thalassolituus oleivorans IDA Negative Gram stain IDA Curved rods Cell shape IDA Motile Motility IDA Non-sporulating Sporulation IDA 4-32°C Temperature range IDA 25°C Optimum temperature IDA Sodium acetate, Tween-40, Tween-80, Carbon source alkanes (C12-C36) IDA Chemoorganotrophic Energy source IDA Oxygen Terminal electron receptor IDA Surface seawater MIGS-6 Habitat IDA 0.5-5% NaCl (w/v) MIGS-6.3 Salinity IDA Aerobic MIGS-22 Oxygen IDA Free-living MIGS-15 Biotic relationship IDA Unknown MIGS-14 Pathogenicity NAS Chukchi Sea, Arctic Ocean MIGS-4 Geographic location IDA July 2010 MIGS-5 Sample collection time IDA 69°30.00′ MIGS-4.1 Latitude IDA MIGS-4.2
Longitude
MIGS-4.3
Depth
MIGS-4.4
Altitude
-168°59.00′
Surface seawater Sea level
IDA IDA IDA
a) Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [20]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.
When compared to other Thalassolituus species, strain R6-15 differed from type strain MIL-1T [1] in catalase, urease and acid phosphatase, and in the utilization of n-alkane, pyruvic acid methyl ester, D-mannitol and D-sorbitol (Table 2). Differences were also observed with type strain http://standardsingenomics.org
IMCC1826T [2] in growth temperature range, catalase, nitrate reductase, urease and leucine arylamidase and the utilization of n-alkane, pyruvic acid methyl ester, β-Hydroxybutyric acid and D,LLactic acid (Table 2). 895
Thalassolituus oleivorans Table 2. Differential phenotypic characteristics between T. oleivorans R6-15 and other Thalassolituus species. Characteristic Cell diameter (µm) Salinity/Optimum (w/v) Temperature range (°C) Number of polar flagella Production of Catalase Nitrate reductase Urease Acid phosphatase Leucine arylamidase Carbon source Sodium acetate n-alkane Pyruvic acid methyl ester β -Hydroxybutyric acid D,L-Lactic acid D-Mannitol D-Sorbitol
1 0.25-0.4 x 1.2-2.0 0.5-5%/ 3% 4-32 1
2 0.32-0.77x1.2-3.1 0.5-5.7%/ 2.3% 4-30 1-4
3 0.4-0.5 x1.2-2.5 0.5-5.0%/ 2.5% 15-42 1
w + +
+ +
+ + + + -
+ + na C12-C36 C7-C20 C14 and C16 w + + + + + Chukchi Sea, Harbor of Milazzo, Deokjeok island, KoGeographic location Arctic Ocean Italy rea Habitat surface seawater seawater/sediment surface seawater G+C content (mol%) 46.6 46.6 54.6 Strains: 1, T. oleivorans R6-15; 2, T. oleivorans MIL-1T; 3, T. marinus IMCC1826T. +: positive result, -: negative result, w: weak positive result, na: data not available.
Genome sequencing information Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position and dominance position in the crude oil-degrading consortia enriched from the surface seawater of the Arctic Ocean. The complete genome sequence was deposited in Genbank under accession number Table 3. Project information MIGS ID Property
CP006829. Sequencing, finishing and annotation of the T. oleivorans R6-15 genome were performed by the Chinese National Human Genome Center (Shanghai). Table 3 presents the project information and its association with MIGS version 2.0 compliance [21]. Term
MIGS-31
Finishing quality
Finished
MIGS-28
Libraries used
one 454 pyrosequence standard library
MIGS-29
Sequencing platforms
454 GS FLX Titanium
MIGS-31.2
Fold coverage
21.1 ×
MIGS-30
Assemblers
Newbler version 2.7
MIGS-32
Gene calling method
NCBI PGAP pipeline
GenBank ID
CP006829
GenBank Date of Release
On publication
GOLD ID
Gi20060
Project relevance
Crude oil-degradation, biogeography
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Growth conditions and DNA isolation
Strain R6-15 was grown aerobically in ONR7a medium [22] with sodium acetate as the sole carbon and energy source. The genomic DNA was extracted from the cell, concentrated and purified using the AxyPrep bacterial genomic DNA miniprep Kit (Axygen), as detailed in the manual for the instrument.
Genome sequencing and assembly
The genome was sequenced by using a massively parallel pyrosequencing technology (454 GS FLX) [23]. A total of 140,550 reads counting up to 78,223,504 bases were obtained, covered 21.1folds of genome. The Newbler V2.7 [24] software package was used for sequence assembly and quality assessment. After assembling, 64 contigs ranging from 500 bp to 304,980 bp were obtained, and the relationship of the contigs was determined by multiplex PCR [25]. Gaps were then filled in by sequencing the PCR products using ABI 3730xl capillary sequencers. A total of 284 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Finally, the sequences were assembled using Phred, Phrap and Consed software packages [26], and low quality regions of the genome were resequenced. The final sequence accuracy was approximately 99.999%. Table 4. Genome statistics Attribute Genome size (bp) DNA coding region (bp) DNA G+C content (bp) Number of replicons Extrachromosomal elements Total genes RNA genes tRNA genes rRNA operons ncRNA genes Protein-coding genes Pseudo genes Genes with function prediction Genes in paralog clusters Genes assigned to COGs Genes assigned Pfam domains Genes with signal peptides Genes with transmembrane helices
Dong et al.
Genome annotation
The protein-coding genes, structural RNAs (5S, 16S, 23S), tRNAs and small non-coding RNAs were predicted and achieved by using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) server online [27]. The functional annotation of predicted ORFs was performed using RPS-BLAST [28] against the cluster of orthologous groups (COG) database [29] and Pfam database [30]. TMHMM program was used for gene prediction with transmembrane helices [31] and signalP program was used for prediction of genes with peptide signals [32].
Genome properties
The properties and the statistics of the genome are summarized in Table 4. The genome includes one circular chromosome of 3,764,053 bp (46.6% GC content). In total, 3,489 genes were predicted, 3,372 of which are protein-coding genes, and 61 RNAs; 56 pseudogenes were also identified. The majority of the protein-coding genes (67.07%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 5 and Figure 3. Value 3,764,053 3,315,444 1,753,947 1 0 3,489 61 48 4 1 3,372 56 2,340 1,051 2,249 2,576 338 775
% of Totala 100.0 88.08 46.60 100.00 1.75 1.38 0.03 96.65 1.61 67.07 30.12 64.46 73.83 9.69 22.21
The total is based on either the size of the genome in base pairs or on the total number of protein coding genes in the annotated genome. a
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Thalassolituus oleivorans
Figure 3. Graphical map of the chromosome. From outside to the center: Genes on forward strand (color by COG categories), genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red), GC content, GC skew. Table 5. Number of genes associated with the 25 general COG functional categories Code Value %age Description J 182 7.11 Translation, ribosomal structure and biogenesis A 1 0.04 RNA processing and modification K 161 6.29 Transcription L 132 5.16 Replication, recombination and repair B 1 0.04 Chromatin structure and dynamics D 32 1.25 Cell cycle control, cell division, chromosome partitioning Y 0 0.00 Nuclear structure V 28 1.09 Defense mechanisms T 152 5.94 Signal transduction mechanisms M 150 5.86 Cell wall/membrane/envelope biogenesis N 85 3.32 Cell motility Z 1 0.04 Cytoskeleton W 0 0.00 Extracellular structures U 83 3.24 Intracellular trafficking, secretion, and vesicular transport O 127 4.96 Posttranslational modification, protein turnover, chaperones C 143 5.59 Energy production and conversion G 76 2.97 Carbohydrate transport and metabolism E 187 7.30 Amino acid transport and metabolism F 67 2.62 Nucleotide transport and metabolism H 115 4.49 Coenzyme transport and metabolism I 106 4.14 Lipid transport and metabolism P 138 5.39 Inorganic ion transport and metabolism Q 57 2.23 Secondary metabolites biosynthesis, transport and catabolism R 329 12.85 General function prediction only S 207 8.09 Function unknown 1240 35.54 Not in COGs 898
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Dong et al.
Insights from the genome sequence Until now, only the genome sequence of the type strain T. oleivorans MIL-1T was available within the genus of Thalassolituus [9]. Here, we compared the genome of strain R6-15 with strain MIL1T (Table 6). The genome of strain R6-15 is nearly 156 kb smaller in size than strain MIL-1T. The G+C content of strain R6-15 (46.6%) is similar with type strain MIL-1T (46.6%). The gene content of strain R6-15 is smaller than strain MIL-1T (3,489 vs 3,732). Strain R6-15 shares 2,995 orthologous genes with type strain MIL-1T. The average percentage of nu-
cleotide sequence identity is 96.92% between strain R6-15 and MIL-1T. In addition, DNA-DNA hybridization (DDH) estimate value between strain R6-15 and MIL-1T were calculated using the genome-to-genome distance calculator (GGDC2.0) [33,34]. The DDH estimate value between them was 84.5% ± 2.57, which were above the standard criteria (70%) [35]. Therefore, these results confirmed that strain R6-15 belonged to the species of Thalassolituus oleivorans.
ProProtein Genome Genome Gene tein with Name size (bp) count coding function T. oleivorans R6-15 3,764,053 3,489 3,372 2,340 T. oleivorans MIL-1T 3,920,328 3,732 3,603 2,038 Table 6. Comparison of genomes between T. oleivorans R6-15 and T. oleivorans MIL-1T
Conclusion
Strain R6-15 is the first strain with the complete genome sequence of the genus Thalassolituus isolated from the Arctic Ocean. These genomic data will provide insights into the mechanisms of how this bacterium can thrive on the crude oil in the polar marine environments.
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DOI:10.4056/sigs.5229330
Complete genome sequence of Thalassolituus oleivorans R6-15, an obligate hydrocarbonoclastic marine bacterium from the Arctic Ocean Chunming Dong1, 2, 3†, Xin Chen1, 2, 3, 4†, Yanrong Xie1, 2, 3, 4, Qiliang Lai1, 2, 3, Zongze Shao1,2, 3* Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, China; 2 State Key Laboratory Breeding Base of Marine Genetic Resources, Xiamen, China; 3 Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen, China; . 4 Life Science College, Xiamen University, Xiamen 361005, Fujian, China. 1
Correspondence: Zongze Shao ([email protected])
*
†
Authors contributed equally to this work.
Keywords: Thalassolituus, genome, alkane-degrading, surface seawater, Arctic Ocean Strain R6-15 belongs to the genus Thalassolituus, in the family Oceanospirillaceae of Gammaproteobacteria. Representatives of this genus are known to be the obligate hydrocarbonoclastic marine bacteria. Thalassolituus oleivorans R6-15 is of special interest due to its dominance in the crude oildegrading consortia enriched from the surface seawater of the Arctic Ocean. Here we describe the complete genome sequence and annotation of this strain, together with its phenotypic characteristics. The genome with size of 3,764,053 bp comprises one chromosome without any plasmids, and contains 3,372 protein-coding and 61 RNA genes, including 12 rRNA genes.
Introduction
Thalassolituus spp. belong to the Oceanospirillaceae of Gammaproteobacteria. The genus was first described by Yakimov et.al. (2004), and is currently composed of two type species, T. oleivorans and T. marinus [1,2]. Bacteria of this genus are known as obligate hydrocarbonoclastic marine bacteria [3]. Previous reports showed that Thalassolituusrelated species were among the most dominant members of the petroleum hydrocarbon-enriched consortia at low temperature [4-7]. In addition to consortia enriched with oil, Thalassolituus spp. can be detected in variety of cold environments as well [8-10]. Strain R6-15 was isolated from the surface seawater of the Arctic Ocean after enriched with crude oil during the fourth Chinese National Arctic Research Expedition of the “Xulong” icebreaker in the summer of 2010. The 16S rRNA gene sequence shared 99.86% and 96.39% similarities with T. oleivorans MIL-1T and T. marinus IMCC1826T, respectively. Pyrosequencing results (16S rRNA gene V3 region) of fifteen oil-degrading consortia across the Arctic Ocean showed that the dominant
member in most of the consortia shared identical sequence of this strain, comprising 8.4-99.6% of the total reads (not published). Here, we described the complete genome sequence and annotation of strain T. oleivorans R615, and its phenotypic characteristics. Moreover, a brief comparison was made between strain R6-15 and the two type strains of the validly named species of this genus, in both phenotypic and genomic aspects.
Classification and features
T. oleivorans R6-15 is closely related with T. oleivorans MIL-1T (Figure 1, Table 1). The strain is aerobic, Gram-negative and motile by a single polar flagellum, exhibiting a characteristic morphology of a curved rod-shape cell (Figure 2). Strain R6-15 is able to utilize a restricted spectrum of carbon substrates for growth, including sodium acetate, Tween-40, Tween-80 and C12-C36 aliphatic hydrocarbons. Its growth temperature ranges from 4 to 32°C with optimum of 25°C.
The Genomic Standards Consortium
Thalassolituus oleivorans 10 100
Thalassolituus oleivorans MIL-1T (AJ431699) Thalassolituus oleivorans R6-15 (KF836058)
89
Oceanobacter kriegii IFO 15467T (AB006767) Bermanella marisrubri RED65T (AY136131)
95
Marinomonas blandensis MED 121 (DQ403809) Marinomonas mediterranea MMB-1T (AF063027)
100
Marinomonas sp. MWYL1 (NR_074778)
85 6 100
Marinomonas posidonica IVIA-Po-181T (EU188445) Oceanospirillum beijerinckii IFO 15445T (AB006760) Oceanospirillum maris IFO 15468T (AB006763)
Neptunomonas japonica JAMM 0745T (AB288092) Amphritea japonica JAMM 1866T (AB330881)
63 7
Neptuniibacter caesariensis MED92T (AY136116) Marinospirillum minutulum ATCC 19193T 0.0
Figure 1. Phylogenetic tree highlighting the position of T. oleivorans strain R6-15 relative to other type and nontype strains with finished or non-contiguous finished genome sequences within the family Oceanospirillaceae. Accession numbers of 16S rRNA gene sequences are indicated in brackets. Sequences were aligned using DNAMAN version 6.0, and a neighbor-joining tree obtained using the maximum-likelihood method within the MEGA version 5.0 [11]. Numbers adjacent to the branches represent percentage bootstrap values based on 1,000 replicates.
Figure 2. Transmission electron micrograph of T. oleivorans R6-15, using a JEM-1230 (JEOL) at an operating voltage of 120 kV. The scale bar represents 0.5 µm. 894
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Dong et al. Table 1. Classification and general features of T. oleivorans R6-15 according to the MIGS recommendations [12]. MIGS ID Property Term Evidence codea Domain Bacteria TAS [13] Phylum Proteobacteria TAS [14] Class Gammaproteobacteria TAS [15-17] Order Oceanospirillales TAS [16,18] Current classification Family Oceanospirillaceae TAS [16,19] Genus Thalassolituus TAS [1] Species Thalassolituus oleivorans IDA Negative Gram stain IDA Curved rods Cell shape IDA Motile Motility IDA Non-sporulating Sporulation IDA 4-32°C Temperature range IDA 25°C Optimum temperature IDA Sodium acetate, Tween-40, Tween-80, Carbon source alkanes (C12-C36) IDA Chemoorganotrophic Energy source IDA Oxygen Terminal electron receptor IDA Surface seawater MIGS-6 Habitat IDA 0.5-5% NaCl (w/v) MIGS-6.3 Salinity IDA Aerobic MIGS-22 Oxygen IDA Free-living MIGS-15 Biotic relationship IDA Unknown MIGS-14 Pathogenicity NAS Chukchi Sea, Arctic Ocean MIGS-4 Geographic location IDA July 2010 MIGS-5 Sample collection time IDA 69°30.00′ MIGS-4.1 Latitude IDA MIGS-4.2
Longitude
MIGS-4.3
Depth
MIGS-4.4
Altitude
-168°59.00′
Surface seawater Sea level
IDA IDA IDA
a) Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [20]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.
When compared to other Thalassolituus species, strain R6-15 differed from type strain MIL-1T [1] in catalase, urease and acid phosphatase, and in the utilization of n-alkane, pyruvic acid methyl ester, D-mannitol and D-sorbitol (Table 2). Differences were also observed with type strain http://standardsingenomics.org
IMCC1826T [2] in growth temperature range, catalase, nitrate reductase, urease and leucine arylamidase and the utilization of n-alkane, pyruvic acid methyl ester, β-Hydroxybutyric acid and D,LLactic acid (Table 2). 895
Thalassolituus oleivorans Table 2. Differential phenotypic characteristics between T. oleivorans R6-15 and other Thalassolituus species. Characteristic Cell diameter (µm) Salinity/Optimum (w/v) Temperature range (°C) Number of polar flagella Production of Catalase Nitrate reductase Urease Acid phosphatase Leucine arylamidase Carbon source Sodium acetate n-alkane Pyruvic acid methyl ester β -Hydroxybutyric acid D,L-Lactic acid D-Mannitol D-Sorbitol
1 0.25-0.4 x 1.2-2.0 0.5-5%/ 3% 4-32 1
2 0.32-0.77x1.2-3.1 0.5-5.7%/ 2.3% 4-30 1-4
3 0.4-0.5 x1.2-2.5 0.5-5.0%/ 2.5% 15-42 1
w + +
+ +
+ + + + -
+ + na C12-C36 C7-C20 C14 and C16 w + + + + + Chukchi Sea, Harbor of Milazzo, Deokjeok island, KoGeographic location Arctic Ocean Italy rea Habitat surface seawater seawater/sediment surface seawater G+C content (mol%) 46.6 46.6 54.6 Strains: 1, T. oleivorans R6-15; 2, T. oleivorans MIL-1T; 3, T. marinus IMCC1826T. +: positive result, -: negative result, w: weak positive result, na: data not available.
Genome sequencing information Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position and dominance position in the crude oil-degrading consortia enriched from the surface seawater of the Arctic Ocean. The complete genome sequence was deposited in Genbank under accession number Table 3. Project information MIGS ID Property
CP006829. Sequencing, finishing and annotation of the T. oleivorans R6-15 genome were performed by the Chinese National Human Genome Center (Shanghai). Table 3 presents the project information and its association with MIGS version 2.0 compliance [21]. Term
MIGS-31
Finishing quality
Finished
MIGS-28
Libraries used
one 454 pyrosequence standard library
MIGS-29
Sequencing platforms
454 GS FLX Titanium
MIGS-31.2
Fold coverage
21.1 ×
MIGS-30
Assemblers
Newbler version 2.7
MIGS-32
Gene calling method
NCBI PGAP pipeline
GenBank ID
CP006829
GenBank Date of Release
On publication
GOLD ID
Gi20060
Project relevance
Crude oil-degradation, biogeography
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Growth conditions and DNA isolation
Strain R6-15 was grown aerobically in ONR7a medium [22] with sodium acetate as the sole carbon and energy source. The genomic DNA was extracted from the cell, concentrated and purified using the AxyPrep bacterial genomic DNA miniprep Kit (Axygen), as detailed in the manual for the instrument.
Genome sequencing and assembly
The genome was sequenced by using a massively parallel pyrosequencing technology (454 GS FLX) [23]. A total of 140,550 reads counting up to 78,223,504 bases were obtained, covered 21.1folds of genome. The Newbler V2.7 [24] software package was used for sequence assembly and quality assessment. After assembling, 64 contigs ranging from 500 bp to 304,980 bp were obtained, and the relationship of the contigs was determined by multiplex PCR [25]. Gaps were then filled in by sequencing the PCR products using ABI 3730xl capillary sequencers. A total of 284 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Finally, the sequences were assembled using Phred, Phrap and Consed software packages [26], and low quality regions of the genome were resequenced. The final sequence accuracy was approximately 99.999%. Table 4. Genome statistics Attribute Genome size (bp) DNA coding region (bp) DNA G+C content (bp) Number of replicons Extrachromosomal elements Total genes RNA genes tRNA genes rRNA operons ncRNA genes Protein-coding genes Pseudo genes Genes with function prediction Genes in paralog clusters Genes assigned to COGs Genes assigned Pfam domains Genes with signal peptides Genes with transmembrane helices
Dong et al.
Genome annotation
The protein-coding genes, structural RNAs (5S, 16S, 23S), tRNAs and small non-coding RNAs were predicted and achieved by using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) server online [27]. The functional annotation of predicted ORFs was performed using RPS-BLAST [28] against the cluster of orthologous groups (COG) database [29] and Pfam database [30]. TMHMM program was used for gene prediction with transmembrane helices [31] and signalP program was used for prediction of genes with peptide signals [32].
Genome properties
The properties and the statistics of the genome are summarized in Table 4. The genome includes one circular chromosome of 3,764,053 bp (46.6% GC content). In total, 3,489 genes were predicted, 3,372 of which are protein-coding genes, and 61 RNAs; 56 pseudogenes were also identified. The majority of the protein-coding genes (67.07%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 5 and Figure 3. Value 3,764,053 3,315,444 1,753,947 1 0 3,489 61 48 4 1 3,372 56 2,340 1,051 2,249 2,576 338 775
% of Totala 100.0 88.08 46.60 100.00 1.75 1.38 0.03 96.65 1.61 67.07 30.12 64.46 73.83 9.69 22.21
The total is based on either the size of the genome in base pairs or on the total number of protein coding genes in the annotated genome. a
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Thalassolituus oleivorans
Figure 3. Graphical map of the chromosome. From outside to the center: Genes on forward strand (color by COG categories), genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red), GC content, GC skew. Table 5. Number of genes associated with the 25 general COG functional categories Code Value %age Description J 182 7.11 Translation, ribosomal structure and biogenesis A 1 0.04 RNA processing and modification K 161 6.29 Transcription L 132 5.16 Replication, recombination and repair B 1 0.04 Chromatin structure and dynamics D 32 1.25 Cell cycle control, cell division, chromosome partitioning Y 0 0.00 Nuclear structure V 28 1.09 Defense mechanisms T 152 5.94 Signal transduction mechanisms M 150 5.86 Cell wall/membrane/envelope biogenesis N 85 3.32 Cell motility Z 1 0.04 Cytoskeleton W 0 0.00 Extracellular structures U 83 3.24 Intracellular trafficking, secretion, and vesicular transport O 127 4.96 Posttranslational modification, protein turnover, chaperones C 143 5.59 Energy production and conversion G 76 2.97 Carbohydrate transport and metabolism E 187 7.30 Amino acid transport and metabolism F 67 2.62 Nucleotide transport and metabolism H 115 4.49 Coenzyme transport and metabolism I 106 4.14 Lipid transport and metabolism P 138 5.39 Inorganic ion transport and metabolism Q 57 2.23 Secondary metabolites biosynthesis, transport and catabolism R 329 12.85 General function prediction only S 207 8.09 Function unknown 1240 35.54 Not in COGs 898
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Dong et al.
Insights from the genome sequence Until now, only the genome sequence of the type strain T. oleivorans MIL-1T was available within the genus of Thalassolituus [9]. Here, we compared the genome of strain R6-15 with strain MIL1T (Table 6). The genome of strain R6-15 is nearly 156 kb smaller in size than strain MIL-1T. The G+C content of strain R6-15 (46.6%) is similar with type strain MIL-1T (46.6%). The gene content of strain R6-15 is smaller than strain MIL-1T (3,489 vs 3,732). Strain R6-15 shares 2,995 orthologous genes with type strain MIL-1T. The average percentage of nu-
cleotide sequence identity is 96.92% between strain R6-15 and MIL-1T. In addition, DNA-DNA hybridization (DDH) estimate value between strain R6-15 and MIL-1T were calculated using the genome-to-genome distance calculator (GGDC2.0) [33,34]. The DDH estimate value between them was 84.5% ± 2.57, which were above the standard criteria (70%) [35]. Therefore, these results confirmed that strain R6-15 belonged to the species of Thalassolituus oleivorans.
ProProtein Genome Genome Gene tein with Name size (bp) count coding function T. oleivorans R6-15 3,764,053 3,489 3,372 2,340 T. oleivorans MIL-1T 3,920,328 3,732 3,603 2,038 Table 6. Comparison of genomes between T. oleivorans R6-15 and T. oleivorans MIL-1T
Conclusion
Strain R6-15 is the first strain with the complete genome sequence of the genus Thalassolituus isolated from the Arctic Ocean. These genomic data will provide insights into the mechanisms of how this bacterium can thrive on the crude oil in the polar marine environments.
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